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Obrador 2023
Obrador 2023
Article
Externally Applied Electromagnetic Fields and Hyperthermia
Irreversibly Damage Cancer Cells
Elena Obrador 1,2 , Ali Jihad-Jebbar 1 , Rosario Salvador-Palmer 1 , Rafael López-Blanch 1,2 ,
María Oriol-Caballo 1,2 , María Paz Moreno-Murciano 2 , Enrique A. Navarro 2,3,4 , Rosa Cibrian 1,2
and José M. Estrela 1,2,5, *
1 Department of Physiology, Faculty of Medicine and Odontology, University of Valencia, 46010 Valencia, Spain;
elena.obrador@uv.es (E.O.); ajijeb@alumni.uv.es (A.J.-J.); rosario.salvador@uv.es (R.S.-P.);
loblanch@alumni.uv.es (R.L.-B.); maria.oriolc@gmail.com (M.O.-C.); rosa.m.cibrian@uv.es (R.C.)
2 Scientia BioTech, 46002 Valencia, Spain; paz.moreno72@gmail.com (M.P.M.-M.);
enrique.navarro@uv.es (E.A.N.)
3 Department of Computer Sciences, Higher Technical School of Engineering, 46100 Burjassot, Spain
4 IRTIC Institute, University of Valencia, 46980 Paterna, Spain
5 Department of Physiology, Faculty of Pharmacy, University of Valencia, 46100 Burjassot, Spain
* Correspondence: jose.m.estrela@uv.es
1. Introduction
The biological effects of non-ionizing electromagnetic fields (EMFs) have been exten-
sively investigated for decades [1]. Based on the guidelines of the International Commission
on non-Ionizing Radiation Protection, “Radiofrequency Electromagnetic Fields” (RF EMFs)
is the term used to describe the part of the electromagnetic spectrum comprising the
frequency range from 100 kHz to 300 GHz (https://www.icnirp.org (accessed on 18 Oc-
tober 2022)). Electric fields result from differences in voltage, whereas magnetic fields
result from the flow of electric current. Tumor-treating fields (TTFs) are a type of on-
cotherapy that uses alternating electric fields of intermediate frequency (∼100–500 kHz)
and low intensity (1–3 V/cm) to disrupt cell division and promote inhibition of cancer
growth [2]. The use of this range of intermediate frequency is based on evidence showing
that low-frequency electric fields (<1 kHz) progressively disrupt cell membrane polar-
ization, whereas high-frequency fields (>1000 kHz) cause heating-induced damages due
to the vibration of the charged/polar cell molecules [3,4]. The established technique for
applying the TTFs requires placing electrodes on the body surface around the focus of
tumor growth so that a potential difference can be generated across growing cancer [5]
(https://www.novocure.com (accessed on 7 April 2022)). Charged molecules, if subjected
to an electric or an EMF, are affected by the direction and intensity of the energy flow.
However, there are key differences in the way electric and magnetic fields interact with
charged molecules, i.e., (but not limited to) (a) the electric field lines are in the direction of
the voltage gradient and do not form a loop, whereas the magnetic field lines are around
the currents forming a closed loop; (b) the electric field is inversely proportional to the
voltage gradient, whereas the intensity of the magnetic field depends on the number of
field lines produces by the magnet and the current enclosed in its loop; (c) the electric field
lines are measured in two dimensions, whereas the magnetic field lines are measured in
three dimensions (https://www.niehs.nih.gov (accessed on 23 January 2023)). At present,
potential applications of non-thermal EMFs on cancer therapy have not been implemented
yet [6].
Molecular vibration increases as temperature increases and hyperthermia (HT) can
damage and kill cancer cells. However, in practice, HT-based applications in oncotherapy
still face strong limitations [7]. These limitations include (a) HT can cause damage to
healthy tissues surrounding the tumor if the temperature is not precisely controlled (e.g.,
burns, blistering, and necrosis); (b) patients may experience pain, discomfort, or a burning
sensation during the HT treatment; (c) HT may cause skin reactions such as redness,
swelling, or rash in the treated area; (d) limited penetration depending on the source and
type of energy applied; (e) the response to HT can vary among individuals and tumor
types (some tumors may be more sensitive to heat, while others may be less responsive);
(f) ensuring precise targeting of the tumor area while avoiding damage to surrounding
healthy tissues is challenging. Thus, advanced imaging techniques and treatment planning
are necessary to improve targeting accuracy. Based on the guidelines of the US National
Cancer Institute, HT is a type of treatment in which body tissue is heated to as high
as 45 ◦ C to help damage and kill cancer cells with little or no harm to normal tissue
(http://www.cancer.gov (accessed on 1 August 2021)). To this end, suggested techniques
include probes that generate energy from microwaves, radio waves, lasers, ultrasounds,
perfusion of heating fluids, or heating of an entire body in a heated chamber or hot
water (http://www.cancer.gov (accessed on 10 October 2022)). All these approaches have
potential side effects and limited efficacy [8].
Although electromagnetic and heat energies may affect many different cell functions
(e.g., [9]), their association is still underdeveloped as a potential oncotherapy. Magnetic
Cancers 2023, 15, 3413 3 of 24
2.2. Experimental Setup for the Combined Treatment with EMFs and HT In Vitro
The source of EMFs was generated with a Promax-GFG-8216 generator (GW Instek,
Taipei, Taiwan), adjusted for an output signal of f (frequency) = 100–500 KHz, with an
amplitude of 2 V. This signal can be visualized with an oscilloscope to check for accuracy
of amplitude and f of the sinusoidal signal. The output of the generator was connected
with a BCN connector to a coaxial cable of 50 Ω of characteristic impedance. The end of it
was soldered to a WE-760308102142 coil from Würth Electronik (Rot am See, Germany).
According to the manufacturer’s information, the coil has an inductance L = 5.8 µH and a
DC resistance (or ohmic resistance of a conductor) of 0.01 Ω. The calculated skin resistance
at, e.g., 100 KHz is 0.014 Ω. The coil consists of two superimposed windings, with two
parallel copper wires forming a 5-winding flat spiral.
The current can be easily calculated based on the generator output voltage and the coil
impedance obtained from the manufacturer, and the magnetic field can be calculated with
this current and its geometrical distribution. The WE-760308102142 coil is mathematically
modeled as two sets of 10 concentric turns of increasing radius, one set above the other. The
outer diameter of the coils was 48.85 mm, and the copper wire had a diameter of 1.5 mm.
The magnetic induction B-field (Tesla) along the axis is calculated as follows:
" #
µ0 I n ak 2 n
ak 2
2 k∑
B= +∑
2 3/2 2 3/2
=1 a 2 + z
k 1 k =0 a 2 + z
k 2
where
V
I= q
R2 + ( Lω − 1/Cω )2
Cancers 2023, 15, 3413 4 of 24
2.7. H2 O2 and O2 −
Quantitative measurement of H2 O2 and O2 − generation followed the previously
described methodology [15].
2.9. ATP
ATP levels were measured fluorometrically following standard enzymic methods [17].
2.10. Glutathione
Glutathione (GSH) was determined by LC/MS, as previously reported [18]. Cell
processing was performed according to the published methodology, where rapid N-
ethylmaleimide derivatization was used to prevent GSH auto-oxidation [19].
2.11. Caspase 3
This activity was measured using a highly sensitive colorimetric substrate, N-acetyl-
Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA), following the manufacturer’s instruc-
tions (CalBiochem, La Jolla, CA, USA). Briefly, cancer cells were lysed in lysis buffer [50 mM
HEPES (pH 7.4), 100 mM NaCl, 0.1% (v/v) CHAPS, 1 mM dithiothreitol and 0.1 mM EDTA]
on ice for 10 min, then centrifuged at 10,000× g for 10 min at 4 ◦ C. Equal volumes of
the supernatants were added to equal volumes of assay buffer [50 mM HEPES (pH 7.4),
100 mM NaCl, 0.1% (v/v) CHAPS, 10 mM dithiothreitol, 0.1 mM EDTA, and 10% glycerol]
and incubated at 37 ◦ C for 10 min. Then, freshly prepared Ac-DEVD-pNA (200 mM) was
added to the mixture, and A405 was monitored every 20 min for 3 h at room temperature.
Cultures without cell lysates were used as controls. Enzyme activity was calculated using
the manufacturer’s formulae as pmol/min.
Z-DEVD-FMK (Z-Asp-Glu-Val-Asp-fluoromethylketone; CalBiochem), dissolved in
DMSO and added in a 0.2% volume to give the concentration indicated in Section 3, was
used as an irreversible caspase 3 inhibitor.
Cancers 2023, 15, 3413 6 of 24
no noticeable motion was observed in the real-time monitored ultrasound images of the
tumor. Mice were treated once per day for three consecutive days. In each session, mice
were subjected for 40 min to the combined effect of EMFs and the HIFU-induced HT. The
total energy generated by the two HIFU transducers in the tumor was that corresponding to
a potency of approximately 60 W/cm2 . To make sure that under our experimental condition,
a temperature of approximately 52 ◦ C was reached within the tumor, in previous control
experiments, a thermocouple (TE connectivity, Schaffhausen, Switzerland) was inserted
in different AsPC1 tumors in vivo. Then the tumors were subjected to HIFU radiation
to make sure which potency was necessary to reach the required temperature. The aim
of this protocol was to maximize the anticancer effect as much as possible while taking
into account the limitations of the in vivo model. Before each session in the bath, mice
were pretreated × 12 h with EMFs in animal housing cages. These cages were surrounded
by an attached network of copper cables that allowed the tumor-bearing mice to receive
approximately 25 µT constantly. The aim of this procedure was to try to maximize the effect
of the EMFs on the growing tumor. Gemcitabine (50 mg/kg) was administered 1 h before
each 3-day treatment period (see Section 3).
3. Results
3.1. EMFs and HT Decrease Cancer Cell Viability
As explained in the introduction and based on the experimental setup (see under
Section 2), exposure to EMFs was not associated with an increase in the cell culture temper-
ature above 37 ◦ C. On the other hand, the protocol to generate HT under in vitro conditions
was designed thinking of its potential application in vivo. An eventual in vivo approach to
increase the tumor temperature should be a) rapid and specifically focused on the tumor
and b) on a scale of temperature that should cause limited damage to normal tissues near
the tumor. To this end, we subjected the cancer cells to a range of temperatures from
37 ◦ C to a maximum of 52 ◦ C. This range of temperature is easy to reach with different
methodologies and (if correctly focused on the tumor) should cause limited side effects in
normal peritumoral tissues. As a proof of concept, we inoculated subcutaneously, AsPC1
cancer cells into nude immunodeficient mice (n = 5) [22]. Two weeks after inoculation, the
tumor volume reached 75–100 mm3 , and it was heated in vivo for 20 or 40 min with an
experimental HIFU device bearing a single transducer (Holosonic S.L., Valencia, Spain).
We observed that (a) the internal temperature of the animal (controlled by a thermal probe
placed in the rectum) remained below 38 ◦ C, whereas (b) the temperature of the tumor
(controlled by a probe placed on the peritumoral skin) could be increased up to 52 ◦ C in less
than 1 min. The spatial shape of the HIFU beam was Gaussian, providing enough accuracy
in heating the tumor; therefore, only the skin surrounding the tumor showed inflammation
(grade 2 after 20 min and grade 3 after 40 min of HIFU treatment). Inflammation was scored
Cancers 2023, 15, 3413 8 of 24
Figure 1. Effect of EMFs and HT on cancer cell viability. (A) Effect of EMFs. Cancer cells were seeded
and 24 h later exposed to EMFs (100–500 kHz × 1–5 h). Control values (0 kHz) were 1.14 ± 0.03
A2058, 1.13 ± 0.04 AsPC1, and 1.20 ± 0.05 MDA-MB-231 (×106 ) viable cells (n = 5 in all cases).
* p < 0.05 comparing all conditions versus controls (0 kHz) (n = 5 t-test). (B) Effect of HT. Cancer cells
were seeded and 24 h later exposed to HT (42–52 ◦ C × 20–40 min). * p < 0.05 ** p < 0.01 comparing all
conditions versus controls (37 ◦ C) ++ p < 0.01 comparing 40 min versus 20 min (n = 5 t test). (C) Effect
of EMFS and HT. Cancer cells were seeded and 24 h later exposed to EMFs (100 kHz × 4 h) and HT
(52 ◦ C × 40 min from min 120 to min 160 of the 4 h period where cells were constantly exposed to
the EMFs). The surviving cells were cultured for 24 additional hours without further exposure to
EMFs and HT. A two-way analysis of variance (ANOVA) was used to make comparisons among
the different groups after 4 h of treatment with EMFs + HT and 24 h after. Different letters indicate
differences p < 0.05 (n = 5).
Cancers 2023, 15, 3413 10 of 24
Figure 2. Cont.
Cancers 2023, 15, 3413 11 of 24
Figure 2. Effect of EMFs and HT on cell cycle distribution and the type of death in cancer cells.
(A) Flow cytometry analysis of the cell cycle distribution after exposure to EMFs and HT as in
Figure 1C (n = 5). No statistically significant differences were found when comparing treatment
with EMFs + HT and controls. (B) Flow cytometry analysis of EMFs and HT-induced apoptosis and
necrosis (treatment as in Figure 1C). A two-way analysis of variance (ANOVA) was used to make
comparisons among control cells treated with EMFs + HT and the different cell subpopulations in
both groups. Different letters indicate differences p < 0.05 (n = 5).
Cancers 2023, 15, 3413 12 of 24
Figure 3. Effect of EMFs and HT on the activation of apoptotic death in cancer cells. (A) Inverted
microscope images (magnification ×10) of cancer cells treated with EMFs and HT (as in Figure 1C)
showing the drastic morphological changes associated with the loss of viability. (B) Cell death analysis
after the 4 h protocol (Figure 1C) based on Hoechst 33342 and propidium iodide (PI) staining and the
TUNEL labeling assay (see under Methods). Cell viability in control flasks was > 98% in all cases. A
one-way analysis of variance (ANOVA) was used to make comparisons among cell subsets. Different
letters indicate statistical differences p < 0.05 (n = 5). (C) Western blots for detection of cytochrome C
and AIF in the cytosolic fraction (all performed right after the 4 h protocol Figure 1C). Densitometric
analysis (a.u. arbitrary units) represents the mean values ± SD for 5 different experiments per cell
line [* p < 0.01 comparing cells treated with EMFs and HT (4 h as in Figure 1C) versus untreated
controls t-test]. The original western blot figures could be found in Figure S3.
3.2. EMFs and HT Increase ROS Generation and the Release of Death Signals from Mitochondria
The induction of cell death by EMFs and HT was further analyzed. We focused our
experiments on combining EMFs and HT because of their superior effect on cancer cell
viability. As shown in Table 1, treatment with EMFs and HT increases O2 consumption and
generation of reactive oxygen species (ROS) in the cancer cells. These effects are associated
with a decrease in mitochondrial membrane potential (MMP), glutathione (mtGSH), and
ATP (mtATP) [all consequences of the damage caused by the increase in ROS [23]]; and
also to an increase in the cytosolic caspase 3 activity (Table 1), AIF and cytochrome C
Cancers 2023, 15, 3413 13 of 24
(Figure 3B) [key executioners of apoptosis [24]]. These experimental facts prove that EMFs
and HT activate the molecular mechanisms of mitochondria-dependent apoptotic death.
It is known that inhibition of cellular energy production, generation of ROS, imbalance
of cellular Ca2+ homeostasis, or extracellular cell death signals are all stimuli capable of
inducing either apoptosis or necrosis. The relative rate of these two processes (protease and
endonuclease activation versus bioenergetic catastrophe) determines whether a cell will
undergo primary necrosis or apoptosis [24], a fact that usually resembles the heterogeneity
of a growing cancer cell population. Despite this, EMFs and HT mainly cause cancer cell
death by apoptosis, whereas only a small % corresponds to necrosis (Figure 2B). From here,
it remains to be elucidated whether the activation of cell death is a direct consequence of
the action of NIR on mitochondria or secondary to other mechanism(s).
Table 1. Effect of EMFs and HT on ROS generation and the molecular mechanisms of apoptosis.
Figure 4. Effect of EMFs and HT on HSP70 and HSP110 and lysosomal permeability. (A) Protein
levels (western blots) of Hsp70 and Hsp110 were measured in cancer cells after exposure to EMFs and
HT (4 h protocol as in Figure 1C) and 24 h after exposure. Densitometric analysis (a.u. arbitrary units)
represents the mean values ± SD for four different experiments per cell line and time point. A one-
way analysis of variance (ANOVA) was used to make comparisons among the different experimental
times. Different letters indicate statistical differences p < 0.05. (B) Cysteine and aspartate cathepsin
activities in the cytosolic fraction were measured after exposure to EMFs and HT (4 h protocol as
in Figure 1C) (n = 5 * p < 0.01 comparing EMFs and HT-treated cells versus untreated controls).
(C) Lysosome staining (LysoTracker) was performed in the cancer cells (representative images) after
the 4 h protocol (as in Figure 1C), showing EMFs and HT-induced diffusion of the lysosomal marker
into the cytosol. The original western blot figures could be found in Figure S3.
As shown in Figure 4B, treatment with EMFs and HT (4 h protocol, as in Figure 1C)
causes an increase in cytosolic cathepsin activities (see also Figure 4C showing lysosomal
content diffusing into the cytosol), thus suggesting that this increase could be the underlying
mechanism activating the mitochondria-dependent apoptotic cells death (Figure 2B). To
Cancers 2023, 15, 3413 15 of 24
prove this hypothesis, we silenced Hsp70 expression in AsPC1 cells before subjecting
them to the effect of EMFs and HT. We used these cells as a proof of concept because
of their relative resistance to EMFs + HT in the short-term (4 h) (Figure 1C). EMFs and
HT (4 h protocol, as in Figure 1C) caused a decrease in wild-type AsPC1 cell viability
to approximately 47% of control values (necrotic cells, based on microscopic analysis,
were <4% of non-viable cells). The same EMFS and HT-induced stress in Hsp70-knockout
(shRNA-dependent) AsPC1 cells drastically decreased viability to approximately 5% of
control values (% of apoptotic and necrotic cells, based on microscopic analysis, was of
approximately 27 and 73% of non-viable cells, respectively) (p < 0.01 comparing Hsp70-
knockout versus wild-type AsPC1 cells, n = 5). The Supplementary Materials Figure S1
shows that, in AsPC1 cells treated with EMFs and HT, the activity of cytosolic cathepsins
further increases in the Hsp70-knockout cell subset. The higher % of necrotic cells in Hsp70-
knockout AsPC1 cells is unsurprising since massive LMP typically results in subapoptotic
or necrotic cell death [30]. These data suggest a direct relationship between EMFs and HT-
induced LMP, Hsp70, cathepsins, and the activation of mitochondria-dependent cell death.
3.4. Strategies to Complement the Anticancer Effect of EMFs and HT and Facilitate the Complete
Elimination of Cancer Cells
The main goal of any anticancer strategy is to eliminate all growing cancer cells
completely. As shown in Figure 1C, exposure to EMFs and HT does not kill all cancer
cells in our experimental conditions. In this, the marked resistance of malignant cells to a
temperature as high as 52 ◦ C is striking. Nevertheless, as explained above, the extremely
low cell viability found 24 h after EMFs and HT treatment may well be misleading. Thus, in
order to make our strategy as efficacious as possible, we first investigated the combination
of EMFs and HT with standard chemotherapy currently in use against the types of cancers
assayed [32–34]. As shown in Figure 5, the combination of EMFs, HT (4 h protocol as in
Figure 1C), and paclitaxel (PAC, in A2058 and MDA-MB-231 cells) or gemcitabine (GEM,
in AsPC1 cells) drastically decreased cancer cell viability to approximately 2.6 (A2058),
5.3 (AsPC1), and 1.7 (MDA-MB-231) % of control values. Cell viability was remeasured
24 h after exposure to the combination of EMFs + HT + chemotherapy, and no viable cell
could be found (Figure 5). In this last 24 h period, the culture medium was renewed to
eliminate the presence of PAC or GEM. Both drugs were incubated at concentrations (1 µM
PAC, 25 µM GEM) that reflect bioavailable concentrations measured after their in vivo
administration and during the period used in our in vitro assays.
PAC is a taxane that binds to tubulin, the protein component of microtubules, simulta-
neously promoting their assembly and disassembly to form stable, nonfunctional micro-
tubules. It is chemotherapy used against different types of cancer, including melanoma [35].
PAC is administered at doses of 100–250 mg/m2 IV (24 h infusion) (http://www.cancer.
org (accessed on 5 April 2022)). Taking the lowest dose of 100 mg/m2 , and approx.
1.8 m2 /70 kg in humans, that dose means approximately 2.6 mg/kg × 24 h or 0.107 mg/kg
× h (0.428 mg/kg × 4 h). The water content in the human body is approximately 0.7 L/kg
of body weight. Therefore, a patient will receive approximately 0.612 mg of PAC/L of body
water × 4 h. Since 854 µg of PAC/L are equivalent to 1 µM (just slightly above the concen-
tration expected using the dose of 100 mg/m2 , we used 1 µM PAC in our experiments. In
fact, plasma levels of unbound PAC are close to 1 µM during a period of 4–5 h after an IV
dose of 135 mg/m2 [36].
GEM is a nucleoside analog that works by blocking the synthesis of new DNA, which
results in cell death. It is a chemotherapy used to treat various carcinomas and as a first-line
treatment alone for pancreatic cancer [37]. GEM is usually administered at 1000 mg/m2 IV
infusions over 30 min once weekly × 7 weeks (http://www.cancer.org (accessed on 5 April
2022)). That means 25.7 mg/kg × 30 min or 36.7 mg of GEM/L of body water × 30 min.
Although 263 µg of GEM/L is equivalent to approximately 139 µM, the mean GEM plasma
concentrations range around 32 µM 30 min after a dose of 1000 mg/m2 , 8 µM at 60 min,
and undetectable levels at 120 min [38]. Based on this pharmacokinetics, we calculated a
Cancers 2023, 15, 3413 16 of 24
Figure 5. Effect of EMFs HT and chemotherapy on cancer cell viability. Cancer cells were seeded 24 h
before starting the treatments. Cells were treated with EMFs and HT (4 h), as in Figure 1C. Paclitaxel
(PAC 1 µM) was present in the cultured medium during the 4 h protocol (Figure 1C). Gemcitabine
(GEM 25 µM) was present in the cultured medium during the last 30 min of the 4 h protocol. At the
end of the 4 h treatment period, the culture medium was changed, and the cells were kept in culture
for 24 additional hours. A two-way analysis of variance (ANOVA) was used to make comparisons
among the different groups after 4 h of treatment with EMFs + HT and 24 h after. Different letters
indicate statistical differences p < 0.05 (n = 5).
Based on the EMFs and HT-induced LMP effect (see above), alternatively, we also
investigated if molecules capable of increasing the LMP could improve the anticancer effect
of EMFs and HT. To this aim, we assayed first a natural polyphenol, pterostilbene (PT),
which has demonstrated LMP properties as well as other anticancer effects [39]. As shown
in Figure 6, PT (20 µM × 4 h) alone very slightly decreased the number of viable cancer
cells as compared to control values. The concentration of this polyphenol was selected
based on pharmacokinetic criteria [40]. PT can be administered in vivo in the form of the
disodium salt of PT phosphate (LGC Standards, Middlesex, UK) to ensure that the selected
concentration of PT can reach the tumor without systemic side effects during a period
equivalent to that used under in vitro conditions (Estrela JM, unpublished observations).
When EMFs and HT (as in Figure 1C) were combined with PT, the number of viable cancer
cells decreased to approximately 7.5 (A2058), 32.7 (AsPC1), and 6.7 (MDA-MB-231) % of
control values (Figure 6).
In addition, we assayed a targeted anti-Hsp70 therapy using apoptozole (Az, N-(4-
carboxamidobenzyl)-2-(3,5-bis-trifluoromethyl)-4,5-bis-(4-methoxyphenyl)-imidazole, Merck).
Az is a small molecule that inhibits the ATPase activity of Hsp70 by binding to its ATPase
domain without affecting other Hsp, and induces apoptotic cancer cell death via caspase
activation [41]. The IC50 values of Az were 4.5 ± 0.3, 5.0 ± 0.5, and 4.0 ± 0.2 µM for A2058,
AsPC1, and MDA-MB-231 cells, respectively (n = 5 in all cases). As shown in Figure 6, Az
alone decreased the number of viable cancer cells to approximately 74 (A2058), 72 (AsPC1),
and 60 (MDA-MB-231) % of control values (Figure 6).
When EMFs and HT (as in Figure 1C) were combined with Az, the number of viable
cancer cells decreased to 0 (A2058), 7.5 (AsPC1), and 0 (MDA-MB-231) % of control values
(Figure 6).
Cancers 2023, 15, 3413 17 of 24
Figure 6. Effect of EMFs HT and a natural lysosomal membrane permeabilizer or a targeted anti-
Hsp70 therapy on cancer cells viability. Cells were treated with EMFs and HT, as in Figure 1C.
Pterostilbene (PT 20 µM) or apoptozole (Az 4–5 µM depending on the IC50 values described in
Section 3) were added to the cultured medium right before (PT) or 12 h before starting the 4 h protocol
(Az) (as in Figure 1C). A one-way analysis of variance (ANOVA) was used to make comparisons
among the different experimental groups. Different letters indicate statistical differences p < 0.05
(n = 5).
3.5. The Combination of EMFs, HT, Standard Chemotherapy, and Pterostilbene Induces a Complete
Regression of Human Pancreatic Cancer Xenografts
As a proof of concept of our therapeutic strategy, we investigated if the combination
of EMFs and HIFU-induced HT could improve the effectiveness of standard chemotherapy
on a human pancreas carcinoma growing in mice. AsPC1 xenografts were treated with
gemcitabine, EMFs, and HIFU-induced HT, or the triple combination. As shown in Figure 7,
gemcitabine, administered at an MTD [42], slightly affected cancer growth (approximately
22% inhibition compared to controls 35 days after inoculation). The effect of treatment with
EMFs and HIFU decreased cancer volume to approximately 14% of controls, whereas the
combination of EMFs + HIFU + gemcitabine decreased cancer volume to approximately
3–4% of controls 35 days after inoculation. Importantly, in our experimental conditions, no
additional side effects were observed when EMFs and HT were added to the chemotherapy.
These results demonstrate the potential efficacy of our strategy. The drastic reduction of
pancreatic cancer growth (Figure 7A) may facilitate its elimination by surgery or by treating
the tumor-bearing animal/patient with an additional targeted therapy (as suggested above).
Based on the data reported in Figure 6, we added PT to the combination of EMFs + HT
+ gemcitabine. A disodium salt of PT phosphate was administered i.p. (as indicated in
the caption of Figure 7). The concentration of PT in the growing tumor was 118 ± 27 µM
30 min after its administration, 59 ± 12 µM (n = 7) at 60 min, and 14 ± 4 µM (n = 7)
at 120 min (n = 7 in all cases). As shown in Figure 7B, complete tumor regression was
achieved using the combination of EMFs + HT + gemcitabine + PT, and no tumor cell
activity was detected using AsPC1 cells transfected with luciferase (Figure 7C). Mice
treated with EMFs + HT + gemcitabine + PT (Figure 7C) were followed up, but tumor
growth activity (assayed once a week with the luciferine-luciferase assay) did not recover
even 2 months after the completion of the treatment. Two months after treatment, all mice
followed health evaluation based on the NIH standard methodology (i.e., animal weight
and complete blood cell count and chemistry) (www.nih.gov (accessed on 14 October 2022)).
Results were similar in control untreated mice and tumor-bearing mice 2 months after the
treatment to eradicate the growing cancer (Supplementary Materials Table S1). Moreover,
data comparing untreated tumor-bearing mice and treated tumor-bearing mice (at day 25,
one day after treatment, see Table S1 and Figure 7A) were not significantly different. This
means that changes in weight, hematology, and clinical chemistry (Table S1) are associated
with the growth of the tumor and are not a consequence of the treatment. Our data indicate
that the therapy is safe and does not compromise key parameters linked to the health status
of mammals.
Cancers 2023, 15, 3413 18 of 24
Figure 7. Effect of EMFs + HIFU-induced HT, gemcitabine and/or PT on the growth of AsPC1
pancreas carcinoma. Cancer cells were inoculated subcutaneously on day 0, and mice were treated
with EMFs and HIFU as described under Materials and Methods. (A) EMFs and HIFU were applied
once per day per three consecutive days (Monday to Wednesday) for two consecutive weeks starting
on day 14 after tumor inoculation. Gemcitabine (50 mg/kg) was administered twice on days 14 and
21. A one-way analysis of variance (ANOVA) was used to make comparisons among the different
experimental groups at each time point. Different letters indicate statistical differences p < 0.05 (n = 15
mice per experimental group). (B) A disodium salt of PT phosphate (Chromadex Inc. Los Angeles
CA) (100 mg of PT/kg) was administered i.p. (one dose 30 min before starting each irradiation session
with EMFs and HT). A one-way analysis of variance (ANOVA) was used to make comparisons among
the different experimental groups. Different letters indicate statistical differences p < 0.05 (n = 12
mice per experimental group). (C) Representative images of mice inoculated with AsPC1/Luciferase
Stable Cells and treated with EMFs HT and gemcitabine (GEM) or EMFs HT gemcitabine and PT.
Cancers 2023, 15, 3413 19 of 24
4. Discussion
Do EMFs cause heating of the cells? The mechanism by which the oscillating magnetic
field may cause heating of the tissue is by inducing Foucault (or Eddy) currents in the
tissue [43]. These currents revolve around the magnetic field lines in the tissue and, by the
Joule effect, could heat the tumor cells. This heating effect is related to the conductivity
(σ) of the living tissue. This conductivity provides the path for microscopic Eddy currents,
which flow in circular paths. The power per unit mass (P) that heats the cells is given by
the following equation: P = π2 ·B2 ·d2 ·f2 /(6·ρ·D). Where B is the magnetic flux density, d is
the depth of tissue over which the magnetic field is provided, f is the field frequency, ρ is
the tissue resistivity (inverse to the electrical conductivity), and D is the mass density of the
tissue [44].
The conductivity of tumor tissue can be up to five times higher than the conductivity
of healthy tissues and has an approximate value of 0.15 Siemens/m at 100–300 kHz [45]. D
for biological tissue is variable (between 900–1050 kg/m3 ) but can be approximated by that
of the water, 1000 kg/m3 [46]. In our experimental setup, the value for p is <20 pW/Kg,
which is very low. Therefore, the mechanism related to the EMF’s effect is not due to
heating. The synergic effect with HT could be attributable, at least in part, to an increase in
the conductivity associated with an increase in the mobility of the charged molecules.
Methods of heating used for cancer treatment involve (a) electromagnetic heating
[i.e., in ascending order of frequency and descending penetration depth, capacitive (using
metal electrodes and 8–25 MHz) or radiative radiofrequency (using extracorporeally placed
antennas with operating frequencies ranging from 60 MHz to 150 MHz), microwave heat-
ing (400–2500 MHz), and infrared (using infrared lamps, frequency > 300 GHz) and laser
heating]; (b) ultrasounds (acoustic energy at frequencies 0.5–10 MHz); (c) hyperthermic
perfusion, generally combined with chemotherapy; (d) conductive heating, as interstitial
implants of metal needles with hot water and palladium–nickel thermoseeds; and (e) mag-
netic nanoparticles, exposed to an external magnetic field (0.1–0.2 MHz) [7]. All these
methods have pros and cons. HIFU refers to intensities >5 W/cm2 , which produce thermal
and mechanical effects, generating a localized temperature increase in the tissues. HIFU
administration allows a precise treatment of targeted areas, where injury to the surrounding
tissue will depend on the temperature reached and the time of exposure. In this regard,
since the ultrasonic energy is focused on a specific volume of tissue, it is key to consider
that the temperature will decrease (based on a Gaussian model) as we move away from that
specific volume. With the purpose of making an approximation of the effects of the two
NIR on a normal cell, we applied our protocol to 3T3 fibroblasts (www.atcc.org (accessed
on 5 July 2021)). As shown in Figure S2A,B, EMFs (100 kHz, as in Figure 1C) or HT at
42 ◦ C (40 min, as in Figure 1C) did not significantly affect 3T3 viability. HT at 47 ◦ C or
52 ◦ C decreased 3T3 viability to approximately 69% and 29%, respectively, of control values
(Figure S2B). As shown in Supplementary Figure S2C, EMFs (100 kHz) and HT at 42 ◦ C did
not decrease 3T3 viability compared to control values. A combination of EMFs (100 kHz)
and HT at 47 ◦ C decreased 3T3 viability to approximately 54% of control values, whereas
EMFs (100 kHz) and HT at 52 ◦ C further decreased 3T3 viability to approximately 15% of
control values (4 h protocol, Figure S2C). Nevertheless, a gradation in the level of damage
dependent on temperature is evident. Hence, it is key to focus the energy that generates
HT in the tumor and use several energy beams, thus preserving as much normal tissue
as possible. In addition, as shown in Figure S2D, EMFs + HT-induced cell death in 3T3
fibroblasts is mainly apoptotic (as in the cancer cells, see Figures 2B and 3B). In unstressed
3T3 fibroblasts, Hsp70 and Hsp110 levels are lower than in the cancer cells, whereas, as
a consequence of their exposure to EMFs and HT (47–52 ◦ C), both Hsp70 and Hsp110
practically disappear (Figure S2E,F). In the case of Hsp70, this is different from what we
observed in cancer cells (Figure 4A), suggesting the need for further studies comparing
cancer cells and their normal counterparts when subjected to the combination of the two
non-ionizing energies.
Cancers 2023, 15, 3413 20 of 24
In practice, the combination of EMFs and HIFU may offer the following advantages:
(a) RF EMFs have not been reported as having any significant toxicity for normal tissues;
(b) magnetic resonance guided-HIFU (MRgFUS) can target specific volumes of cancers (as
small as a few mm of diameter); (c) HIFU can be applied using a fast array program (once
the target is localized, and the volume and shape of the tumor reconstructed in a computer
system, the program can design the sequential application of HIFU at a series of specific
points, thus maximizing efficiency; (d) to work with EMFs and HIFU allows to limit the
rise in temperature to a level that may better preserve the surrounding normal tissues;
(e) although the number of transducers may need to be increased, depending on the location
of the tumor to be treated, a holographic design of the HIFU piezoelectric transducer units
(e.g., patent WO2020/084181A1) can reduce their number and, thus, simplify the system;
(f) the advantage of using multiple transducers is that the energy emitted by each one cannot
damage normal tissues, but it can be concentrated in the growing cancer; (g) if needed,
EMFs and HIFU could be applied several times (e.g., once per day) in order to maximize
their efficacy in vivo. However, in determining the number of energy beams needed to
avoid an excessive rise in temperature in normal tissues (while heating up the cancer to
52 ◦ C), some important issues will have to be considered, i.e., (a) computational models or
simulations that take into account the organ’s geometry, specific location, tissue properties,
and energy propagation characteristics; (b) overlapping beams can cause cumulative
heating effects and potentially raise the temperature beyond the desired limit; (c) the power
and duration of each individual beam must be adjusted to control the amount of energy
delivered to the target and minimize heating in the normal tissues; (d) real-time monitoring
of temperature during the procedure is essential to ensure accurate control; (e) different
tissues have varying thermal conductivities, which impact how heat spreads through them;
(f) the specific absorption rate, or amount of energy absorbed per unit mass of tissue, and
the heat capacity of each organ and tissue. In our procedure, the spatial shape of the HIFU
beam was Gaussian, providing enough accuracy in heating the tumor. Despite this, we
must expect HT-induced damage to the nearest peritumoral tissue. Damage comparable to
the resection of non-cancerous peritumoral tissue that, as a safety margin, is carried out
by surgeons during a resection. Based on the targeting accuracy of ultrasound imaging-
guided robotic HIFU systems for the treatment of solid tumors [47] and the physiological
mechanisms of heat loss in tissues, the temperature a few mm (<5 mm) off target will
drop drastically. Therefore, the combination of EMFs and HIFU can be implemented to
transfer our findings to working in vivo applications (see Figure 7). Moreover, both EMFs
and HIFU are non-invasive techniques that can be further combined with other anticancer
therapies (see under Results). In this regard, the present contribution offers some effective
options. EMFs and HT can be combined with conventional chemotherapy (Figures 5 and 7).
On the other hand, EMFs and HT can also be combined with LMP inducers, such as PT
or a specific anti-Hsp70-targeted drug (Figure 6). At present, PT has been assayed in
clinical trials for different indications, but it has never been administered intravenously to
humans. Interestingly, oral administration of PT cocrystals (as those of PT and picolinic
acid, http://www.circescientific.com (accessed on 14 March 2022)), which increases PT
bioavailability up to 5–10 times compared to that of the natural stilbene alone, can avoid
the need to use the intravenous administration. Moreover, achieving the intratumoral
concentration of the polyphenol is necessary to increase lysosomal permeability. In favor of
the therapeutic use of PT is its potential to decrease Nrf2-dependent antioxidant defenses
in cancer cells [22]. On the other hand, trials involving an anti-Hsp70 targeted therapy are
still in their early beginning. Furthermore, recently, some anticancer lysosomotropic drugs
(e.g., nortriptyline, siramesine, and desipramine) and their nanoformulations have been
engineered to accumulate specifically within these organelles. These drugs can enhance
LMP or disrupt the activity of resident enzymes and protein complexes, such as v-ATPase
and mTORC1 [48] (a list of inducers of lysosomal cell death can be found at, e.g., [49]).
Mechanistically, an increase in cytosolic cathepsin activity triggers the mitochondrial
membrane permeabilization through cleavage of Bid or via activation of phospholipase A2
Cancers 2023, 15, 3413 21 of 24
and the consequent increase in araquidonic acid [50]. Furthermore, cathepsins can directly
cause chromatin condensation [51], whereas the cytosol acidification can lead to L-DNAase
II activation and chromatinolysis [52].
Another interesting aspect that deserves further investigation is the possible role of
mitochondrial HSP in these mechanisms. ROS generation increases with temperature [53]
(Table 1). Thus, it is possible that mitochondrial HSP (Hsp70 in particular), in addition to
their roles in protein transport and folding, protects mitochondrial proteins and DNA from
thermal and ROS damage.
Initially, no reasons may preclude multiple combinations, e.g., EMFs + HT + chemother-
apy + a lysosomal permeabilizer, which is also a feasible option in case of finding an
unexpected cancer cell resistance in vivo. In all this, preclinical studies and clinical trials
will be necessary steps. Naturally, there are no strict restrictions in order to combine EMFs
and HT with other available (or still being implemented) cancer-type-specific therapeutic
options, e.g., immunotherapy or signaling-related targeted therapy. It is also important to
bear in mind the potential counterproductive effects of some lysosomal stabilizers, such as
acetylsalicylic acid or hydrocortisone [54]. These types of drugs should be avoided during
cancer treatment with our strategy.
Despite differences in genetic backgrounds and in vivo behavior among cancer cells,
the combination of EMFS and HT seems to affect them in a similar way (see, e.g., Figure 1C)
and based on the same mechanism (see, e.g., Table 1 and Figure 4). Nevertheless, Hsp70
levels are a clear example of a mechanism of resistance to HT. Hsp70 inhibits the mitochon-
drial outer membrane permeabilization, thus reducing caspase activation and neutralizing
the AIF [55]. Moreover, Hsp70 also localizes to lysosomal membranes and can protect LMP
induced by different stimuli [56]. Therefore, although eventual mechanisms of resistance to
EMFs and HT should be explored in depth, it is encouraging the fact that both energies,
if applied at the correct conditions and time, appear highly effective both in vitro and, as
shown in Figure 7, in vivo.
5. Conclusions
This work demonstrates that a combination of EMFs and HT causes irreversible dam-
age to different cancer cells (i.e., melanoma, pancreatic cancer, and breast cancer). The
mechanism involves permeabilization of the lysosomes, the release of cathepsins to the
cytosol, and activation of the mitochondria-dependent cell death. A combination of EMFs
and HT with standard chemotherapy, molecules that further promote lysosomal perme-
abilization, and/or targeted anti-Hsp70 therapy can completely kill cancer cells. This
strategy, supported by in vitro and in vivo evidence, may complement current oncothera-
pies and can be applied to different cancers. In vivo treated mice followed post-treatment
health evaluation (NIH standard methodology), which showed that in our experimental
conditions, the therapy is safe and, per se, does not compromise mouse physiology.
for the Care and Use of Laboratory Animals NIH Publ. No. 85-23 1985) and approved by the ethics
committee in animal experimentation of the University of Valencia Spain (ref. no. A1454405530866).
Informed Consent Statement: Not applicable.
Data Availability Statement: All raw data regarding these studies are available in the Figshare
repository (https://doi.org/10.6084/m9.figshare.22220065 (accessed on 6 March 2023)).
Acknowledgments: We would like to thank the University of Valencia SCIE (Experimental Research
Support Service) staff for their technical assistance.
Conflicts of Interest: The authors declare that no competing interest or personal relationship have
influenced the work reported in this paper. R. López-Blanch M. Oriol-Caballo and M.P. Moreno-
Murciano receive salary support from ScientiaBiotech.
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